PROJECT SUMMARY This proposal aims to establish DNA cytosine deaminase enzymes as a non-destructive alternative to bisulfite for base-resolution mapping of cytosine modifications. Epigenetic modifications to the genome play an important role in cellular adaptation and in specialization of various cell lineages derived from the same coding sequence. On DNA, these epigenetic changes include modification of cytosine bases at the 5-postion of the nucleobase. The most common modification is 5-methylcytosine (5mC), followed closely by 5- hydroxymethylcytosine (5hmC), a product of TET enzyme-mediated oxidation of 5mC. Transformations involved in development, pluripotency and oncogenesis entail changes in the genomic patterns of 5mC and 5hmC, making it important to have robust methods to localize these modifications. The methods most commonly used to detect these modifications involve treatment of genomic DNA with bisulfite, as the different cytosine modification states have a different propensity for bisulfite-induced deamination which can be analyzed by sequencing. Chemical deamination, however, can degrade the vast majority of starting DNA. As a result, bisulfite-based approaches constrain our ability to understand the landscapes of cytosine modifications in many small or transient cell populations, or to study how changes are coordinated across long stretches of genomic DNA. In this proposal, we will develop and apply DNA deaminase-based sequencing approaches that address the major shortcomings of bisulfite. Our methods rely upon enzymatic, rather than chemical deamination, using APOBEC3A (A3A), a DNA deaminase from the immune system repurposed for these biotechnological applications. In our biochemical studies, we have established that A3A potently discriminates between different cytosine modification states, and, in foundational work leading up to this proposal, we developed APOBEC- Coupled Epigenetic Sequencing (ACE-Seq) as a non-destructive, base resolution sequencing method for localizing 5hmC. Building on this precedent, we propose to advance two new DNA deaminase-based sequencing approaches that can now localize 5mC and 5hmC together, providing a surrogate for bisulfite, or to directly detect 5mC alone through deamination, which is without precedent. We will apply these methods to address important biological questions that are refractory to bisulfite-based approaches, specifically resolving C, 5mC and 5hmC to decipher epigenetic heterogeneity at the single cell level, revealing how in cis changes across loci are coordinated across long stretches of DNA, and reporting on the ?ternary code? of all three modifications in a single read. Our proposal therefore aims to establish DNA deaminases as a non-destructive and more reliable means for sequencing that can displace bisulfite and its associated limitations, and to thereby drive the widespread adoption of DNA deaminases in epigenetic sequencing in the clinic and the lab.